黑曲霉3928植酸酶phyA基因PCR扩增条件的优化     

王喆  宋丽  石星明  倪宏波  徐春厚 《黑龙江畜牧兽医》2007,(10):61-63

根据国内外报道,利用微生物发酵技术生产植酸酶,是获得大量稳定高效植酸酶的主要途径,大量高浓度的植酸酶主要存在于真菌中,但是由于黑曲霉天然菌株产植酸酶酶量低,并且直接使用成本也较高。构建基因工程菌以提高产酶量,进而进行大规模的发酵生产,已成为全球对植酸酶的研究重点。因此,有必要建立直接对植酸酶基因片段有效扩增的相关技术,优化反应条件。聚合酶链式反应(PCR)用于扩增位于两端已知序列之间的DNA区段,自Mullis在1983年建立以来,已成为分子生物学及相关领域的经典试验方法,技术本身也获得长足进步,可靠性不断提高。对PCR扩增…  相似文献   

添加萘乙酸(NAA)对雨生红球藻中虾青素积累的影响     

高政权  孟春晓  车青 《黑龙江畜牧兽医》2007,(11):59-62

近年来,对雨生红球藻合成虾青素几个关键酶基因的转录调控机制也进行了研究,结果表明,β-胡萝卜素酮化酶(bkt)和β-胡萝卜素羟化酶(crtR-B)基因的5′上游侧翼序列中可能存在生长  相似文献   

猪大肠杆菌不耐热肠毒素基因的PCR检测     

陈芳  池仕红  陈汉林  杨林  刘为民  张浩吉  王政富  马春全 《广东畜牧兽医科技》2007,32(6):32-33

以产肠毒素性大肠埃希氏菌(ETEC)不耐热肠毒素(LT)保守序列为靶序列,设计合成了一对可扩增336 bp目的片段的引物,建立了检测ETEC不耐热肠毒素(LT)基因的PCR方法.该方法对987p 、鼠伤寒沙门氏杆菌、猪肺疫巴氏杆菌和链球菌的检测结果均为阴性.对15株分离自腹泻仔猪的待检菌株进行检测,有3株LT基因阳性.结果表明:该方法的特异性和敏感性较高,可用于ETEC的临床快速诊断和流行病学调查.  相似文献   

奶牛α1-抗胰蛋白酶及基因多态性研究进展     

杨海  李秋玲  王力生  王洪梅  李建斌  仲跻峰 《中国畜牧兽医》2007,34(10):47-49

α1-抗胰蛋白酶（α1-antitrypsin，α1AT）是一种多功能糖蛋白。奶牛α1AT编码基因多态性很高，并对奶牛经济性状具有重要意义。作者主要综述了α1-抗胰蛋白酶和其编码基因在奶牛上的研究进展。  相似文献   

山羊催乳素基因PCR-SSCP分析     

赵秀华  储明星  王金玉  方丽  叶素成 《中国畜牧兽医》2007,34(11):35-38

设计5对引物，采用PCR SSCP技术检测催乳素（prolactin，PRL）基因外显子1~外显子5在高繁殖力山羊品种（济宁青山羊、波尔山羊、文登奶山羊）和低繁殖力山羊品种（内蒙古绒山羊）中的单核苷酸多态性，同时研究该基因对济宁青山羊高繁殖力的影响。通过克隆测序首次获得了山羊PRL基因外显子全序列。结果表明PRL基因在研究的4个山羊品种中都不存在单核苷酸多态性，提示检测的PRL基因座位对济宁青山羊高繁殖力没有显著影响。  相似文献   

表达猪瘟兔化疫苗株E0蛋白的PK-15细胞系构建     

亢文华  郝俊峰  潘春刚  宁宜宝 《中国畜牧兽医》2007,34(11):63-65

将克隆到pGEM T-easy载体中的E0基因双酶切回收后，连接到增强型绿色荧光蛋白（EGFP）中，利用Lipofectamine^TM2000将重组子转染PK-15、BHK-21、VERO 3种细胞，直接在荧光显微镜下用蓝光激发进行观察，在3种细胞中都可以观察到绿色荧光，而且在转染后的24、48、72 h 3个时间点上，观察到的荧光细胞数量逐渐增多，在72 h时荧光细胞的数量在3种细胞中都达到最多，总的来看，在PK 15中荧光细胞的数量最多。通过对细胞荧光的定位发现，重组质粒的荧光主要分布在胞浆内，而且细胞核周围的荧光较强，胞核与胞浆的界限明显，尤其是在PK-15细胞和VERO细胞中这种现象尤为明显。未携带E0目的DNA的pEGFP-N1 Vector转染3种细胞后，都可以观察到绿色荧光，但是整个细胞中是均匀出现荧光的。经酶切、PCR、单抗间接免疫荧光检测，PK-E0细胞中有大量的HCLV的E0表达，单纯的PK-15细胞中没有E0的表达。HCLV株在PK-E0细胞中从48h开始到72h的滴度比在PK-15细胞中的滴度要高。证明PK-E0细胞中E0蛋白的大量表达增加了HCLV在细胞中的复制。  相似文献   

Construction of Full-length cDNA of Recombinant Rabies Virus SRV9 Strain Expressing EgM123 Gene of Echinococcus granulosus     

ZHAI Shaohua  ARZUGUL&#  ABLIZ  WANG Nan  HU Meihe  ZHAO Kui  HE Wenqi 《中国畜牧兽医》2007,47(10):3095-3102

The aim of the experiment was to construct the recombinant rabies virus SRV₉ vaccine strain with EgM123 gene by reverse genetics technology and provide the technical means for effective prevention and control of rabies and hydatidosis in China's agricultural and pastoral areas.In this study,the structural protein N,P and L genes of rabies virus SRV₉ were synthesized using gene synthesis technology,which was based on the complete genome sequence of rabies virus SRV₉ and the fusion fragment of the N-P-M fusion fragment and the rabies G gene,through the carrier of enzyme insertion connection methods,the recombinant rabies virus L gene,N-P-M gene fusion fragment and G+EgM123+eGFP gene fusion fragment were successively recombined on the expression vector pcDNA3.1(-) to construct the full-length cDNA of recombinant rabies virus SRV₉ with EgM123 gene.The synthesized genes were constructed on pcDNA3.1(-) expression vector,and the results of transformation,plasmid digestion and gene sequencing showed that the length of N,P,L,N+P+M and G+EgM123+eGFP gene fragments were 1 365,1 107,6 471,3 160 and 3 256 bp,respectively.The full-length cDNA fragment of EgM123 gene recombinant rabies virus full-length cDNA was 12 465 bp,and the sequencing results of each gene fragment were 100%.In this experiment,the full-length cDNA fragment of recombinant EgM123 rabies and eukaryotic expression vectors of the N,P and L genes of rabies virus were successfully constructed,which could save EgM123 gene recombinant rabies by reverse genetics,it also provided the reference for the development of rabies and hydatid disease combined gene recombinant oral live vaccine.  相似文献   

Construction of Brucella ClpS Gene Mutant Strain and Its Effect on Virulence     

LIU Yufu  DONG Hao  SUN Shijing  PENG Xiaowei  CHEN Ruiai  DING Jiabo  JIANG Hui 《中国畜牧兽医》2007,47(11):3767-3773

The present study aimed to determine the role of ClpS gene,and to analyse the impact of ClpS mutation on the virulence of Brucella.A ClpS gene mutant strain,named ΔClpS was constructed by homologous recombination technology.The bacterial growth kinetics,the LPS synthesis ability and the survival ability of bacterial within macrophages as well as the virulence in mouse model were measured.In addition,the difference between parent strain 2308 and the mutant strain ΔClpS were compared.The results showed that under the same culture conditions,no difference in bacterial concentration was observed between 2308 and ΔClpS strains.The silver staining examination showed that the expression level of LPS extracted from two strains were similar,indicating ClpS gene mutation did not alter the growth rate and LPS synthesis ability of Brucella. In the cell infection assay,the survival ability of ΔClpS strain in cells was extremely significantly lower than that of 2308 strain at 72 h after infection (P<0.01).The results of mouse infection experiment showed that in the first week after infection,no significant difference in spleen weight and bacterial concentration between 2308 and ΔClpS strains infected mice was observed.However,at 4 weeks after infection,the bacterial concentration in spleen of ΔClpS infected mice was 10^3.93 CFU/g spleen,which was significantly lower than that of 2308 strain (10^6.68 CFU/g spleen,P<0.01).The spleen weight of ΔClpS infected mice was also remarkably lower than that of 2308 strain (P<0.01).In summary,the results suggested that the ClpS gene of Brucella did not play a role in Brucella growth rate and ability of LPS synthesis,whereas ClpS gene mutation decreased the ability of Brucella colonization in mouse spleen.  相似文献   

Genome-wide Association Study on Alive Litter Size Trait in Bama Xiang Pigs     

MO Jiayuan  GAO Jiuyu  FENG Lingli  LI Yueyue  TIAN Weilong  LIU Xiaoxiao  CHENG Feng  LIANG Liang  LEI Shuqiao  WEN Wei  LIANG Jing  LAN Ganqiu 《中国畜牧兽医》2007,47(12):3965-3975

In order to identify the molecular markers related to alive litter size of Bama Xiang pigs,the genome-wide association study (GWAS) was used to map and screen the candidate genes affecting the alive litter size trait.Ear tissue samples of 297 Bama Xiang pigs with multiple parity records were collected,and DNA was extracted and genotyped by porcine 50K SNP beadchip.After quality control and genotype imputation,the alive litter size of Bama Xiang pigs were GWAS by Tassel.The results showed that the average number born alive per litter of Bama Xiang pigs increased gradually with the increasing of parity in the range of 1-9 parities.A total of 32 816 SNPs were obtained after quality control and filtration.8 SNPs related to alive litter size of Bama Xiang pigs were screened by genome-wide association analysis,which were significant at genome or chromosome level.Based on the enrichment analysis of the coding genes in the region between 500 kb upstream and downstream of the associated significant SNP loci,and the QTL regions and gene functions related to porcine reproductive traits,4 genes (CAPZB,MSH3,CITED2 and HSD17B7) were finally identified to be candidate genes related to alive litter size of Bama Xiang pigs.  相似文献   

禽传染性支气管炎病毒S1基因在毕赤酵母中的表达     

余祖华  王红宁  周生  黄勇  柳萍 《中国兽医学报》2007,27(6):799-802

根据GenBank已公布的传染性支气管炎病毒(Infectious bronchitis virus,IBV)株S1基因序列及pPIC9K表达载体序列,设计1对IBV S1基因表达片段的PCR引物,用RT-PCR方法扩增出长度为1 566 bp IBV S1基因表达片段,5′端不含信号肽序列,3′端添加了终止密码子。用限制性内切酶SnaB和Not将S1基因和载体pPIC9K酶切回收后连接,构建了重组表达载体pPIC9K-S1。用限制性内切酶Bgl将表达质粒pPIC9K-S1线性化,然后用电转化的方法导入毕赤酵母GS115,在MD平板上生长的转化子经过PCR鉴定和表型筛选后,获得了整合型阳性重组菌株GS115/pPIC9K-S1 His Muts。将重组菌株在1%甲醇中进行诱导分泌表达,并对表达产物进行SDS-PAGE、Western blot分析。结果显示,IBV S1基因在毕赤酵母中成功获得了表达,表达蛋白的分子量约为76 000,能与IBV阳性血清特异性结合,表达的蛋白占上清中总蛋白量的12.5%。  相似文献